METHOD 8321B SOLVENT-EXTRACTABLE NONVOLATILE COMPOUNDS BY
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY/THERMOSPRAY/MASS SPECTROMETRY (HPLC/TS/MS) OR ULTRAVIOLET (UV) DETECTION
SW-846 is not intended to be an analytical training manual. Therefore, method
procedures are written based on the assumption that they will be performed by analysts who are formally trained in at least the basic principles of chemical analysis and in the use of the subject technology.
In addition, SW-846 methods, with the exception of required method use for the analysis of method-defined parameters, are intended to be guidance methods which contain general information on how to perform an analytical procedure or technique which a laboratory can use as a basic starting point for generating its own detailed Standard Operating Procedure (SOP), either for its own general use or for a specific project application. The performance data included in this method are for guidance purposes only, and are not intended to be and must not be used as absolute QC acceptance criteria for purposes of laboratory accreditation. 1.0 SCOPE AND APPLICATION
1.1 This method covers the use of high-performance liquid chromatography (HPLC), coupled with both thermospray-mass spectrometry (TS-MS) and an ultraviolet (UV) detector, for the determination of a variety of solvent-extractable nonvolatile compounds, including dyes, organophosphorus compounds, phenoxyacid herbicides, and carbamates. The following compounds have been determined by this method, although not all of the compounds are amenable to UV detection:
Analyte CAS No.a
Azo Dyes Disperse Red 1 2872-52-8 Disperse Red 5 3769-57-1 Disperse Red 13 126038-78-6 Disperse Yellow 5 6439-53-8 Disperse Orange 3 730-40-5 Disperse Orange 30 5261-31-4 Disperse Brown 1 17464-91-4 Solvent Red 3 6535-42-8 Solvent Red 23 85-86-9 Anthraquinone Dyes Disperse Blue 3 2475-46-9 Disperse Blue 14 2475-44-7 Disperse Red 60 17418-58-5
Analyte CAS No.a Organophosphorus Compounds Methomyl 16752-77-5 Thiofanox 39196-18-4 Famphur 52-85-7 Asulam 3337-71-1 Dichlorvos (DDVP) 62-73-7 Dimethoate 60-51-5 Disulfoton 298-04-4 Fensulfothion 115-90-2 Merphos 150-50-5 Parathion methyl 298-00-0 Monocrotophos 6923-22-4 Naled 300-76-5 Phorate 298-02-2 Trichlorfon 52-68-6
Tris (2,3-dibromopropyl) phosphate (Tris-BP) 126-72-7 Chlorinated Phenoxyacid Compounds
Dalapon 75-99-0 Dicamba 1918-00-9 2,4-D 94-75-7 MCPA 94-74-6 MCPP 7085-19-0 Dichlorprop 120-36-5 2,4,5-T 93-76-5 Silvex (2,4,5-TP) 93-72-1 Dinoseb 88-85-7 2,4-DB 94-82-6 2,4-D, butoxyethanol ester 1929-73-3 2,4-D, ethylhexyl ester 1928-43-4 2,4,5-T, butyl ester 93-79-8 2,4,5-T, butoxyethanol ester 2545-59-7 Carbamates Aldicarb* 116-06-3 Aldicarb sulfone 1646-88-4 Aldicarb sulfoxide 1646-87-3 Aminocarb 2032-59-9 Barban 101-27-9 Benomyl 17804-35-2 Bendiocarb* 22781-23-3
Analyte CAS No.a Bromacil 314-40-9 Butylate 2008-41-5 Carbaryl* 63-25-2 Carbendazim* 10605-21-7 Carbofuran* 1563-66-2 Carbosulfan 55285-14-8 Chloropropham 101-21-3 Chloroxuron 1982-47-4
m-Cumenyl methyl carbamate 64-00-6
Diuron* 330-54-1 EPTC 759-94-4 Fenuron 101-42-8 Fluometuron 2164-17-2 Formetanate hydrochloride 23422-53-9 3-Hydroxycarbofuran 16655-82-6 Linuron* 330-55-2 Methiocarb 2032-65-7 Methomyl* 16752-77-5 Metolcarb 1129-41-5 Mexacarbate 315-18-4 Molinate 2212-67-1 Monuron 150-68-5 Neburon 555-37-3 Oxamyl* 23135-22-0 Pebulate 1114-71-2 o-Phenylenediamine 95-54-5 Physostigmine 57-47-6 Physostigmine salicylate 57-64-7 Promecarb 2631-37-0 Propham 122-42-9 Propoxur 114-26-1 Prosulfocarb 52888-80-9 Siduron 1982-49-6 Tebuthiuron 34014-18-1
a Chemical Abstract Service Registry Number.
* These carbamates were tested in a multi-laboratory evaluation. All others were tested in a single-laboratory evaluation.
1.2 The compounds listed in this method were chosen for analysis by HPLC/MS because they have been designated as problem compounds that are hard to analyze by gas chromatographic methods. The sensitivity of this method is dependent upon the level of
interferants within a given matrix, and varies with compound class and even by compound within a class. Additionally, the sensitivity is dependent upon the mode of operation of the mass spectrometer, with the selected reaction monitoring (SRM) mode providing greater sensitivity than single quadrupole scanning.
1.3 This method may be applicable to the analysis of other nonvolatile or semivolatile compounds that are solvent-extractable, are amenable to HPLC, and can be ionized under thermospray introduction for mass spectrometric detection or can be determined by a UV detector. Table 3 lists 10 other classes of compounds that may be amenable to analysis by HPLC/TS/MS.
1.4 This method is designed to detect the chlorinated phenoxyacid compounds (free acid form) and their esters without the use of hydrolysis and esterification in the extraction procedure, although hydrolysis to the acid form is recommended because it will simplify quantitation.
1.5 Data are also provided for the determination of chlorophenoxyacid herbicides in fly ash (Table 12), however, recoveries for most compounds are very low, indicating poor
extraction efficiency for these analytes using the extraction procedure included in this method. Therefore, other extraction procedures, such as those described in the various 3500 series methods for solid samples, should be more effective.
1.6 All of the carbamates and related compounds listed in Sec. 1.1 may be analyzed by HPLC with MS detection. The following carbamates and related compounds also have been evaluated using HPLC with UV detection at two wavelengths. Some compounds can be
determined at both UV wavelengths, and some at only one of the two wavelengths.
Analyte UV/Vis at 254 nm UV/Vis at 280 nm
Barban Y Y Benomyl Y Y Carbaryl Y Y Carbofuran phenol Y Y Carbosulfan Y Y Formetanate hydrochloride Y Y Methiocarb Y Y Mexacarbate Y Y Propham Y Y Thiophanate-methyl Y Y Carbendazim Y Y o-Phenylenediamine Y Y
m-Cumenyl methyl carbamate Y
Oxamyl Y Physostigmine Y Promecarb Y Prosulfocarb Y Thiodicarb Y Triallate Y
Analyte UV/Vis at 254 nm UV/Vis at 280 nm
Bendiocarb Y
Carbofuran Y
Physostigmine salicylate Y
Propoxur Y
1.7 For further compound identification, MS/MS with collision-activated dissociation (CAD) can be used as an optional extension of this method.
1.8 Prior to employing this method, analysts are advised to consult the base method for each type of procedure that may be employed in the overall analysis (e.g., Methods 3500, 3600, 5000, and 8000) for additional information on quality control procedures, development of QC acceptance criteria, calculations, and general guidance. Analysts also should consult the disclaimer statement at the front of the manual and the information in Chapter Two for guidance on the intended flexibility in the choice of methods, apparatus, materials, reagents, and
supplies, and on the responsibilities of the analyst for demonstrating that the techniques employed are appropriate for the analytes of interest, in the matrix of interest, and at the levels of concern.
In addition, analysts and data users are advised that, except where explicitly specified in a regulation, the use of SW-846 methods is not mandatory in response to Federal testing
requirements. The information contained in this method is provided by EPA as guidance to be used by the analyst and the regulated community in making judgments necessary to generate results that meet the data quality objectives for the intended application.
1.9 This method is restricted to use by, or under the supervision of personnel appropriately experienced and trained in the use of high-performance liquid chromatography using mass spectrometers or ultraviolet detectors. Analysts should also be skilled in the interpretation of liquid chromatograms and mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 This method provides reversed-phase high-performance liquid chromatographic (RP/HPLC) and thermospray (TS) mass spectrometric (MS) conditions and ultraviolet (UV) conditions for the detection of the target analytes.
2.1.1 Sample extracts can be analyzed by direct injection into the thermospray or onto a liquid chromatographic-thermospray interface
2.1.2 A gradient elution program is used on the liquid chromatograph to separate the compounds.
2.1.3 Quantitative analysis may be performed by either TS/MS or UV detection, using either an external or internal standard approach. TS/MS detection may be
performed in either a negative ionization (discharge electrode) mode or a positive ionization mode, with a single quadrupole mass spectrometer.
2.1.4 In some cases, the thermospray interface may introduce variability that may cause problems with quantitation. In such instances, the MS response may be used
to identify the analytes of interest while the quantitative results are derived from the response of the UV detector.
2.2 Prior to analysis, appropriate sample preparation techniques must be used. 2.2.1 Samples for analysis of chlorinated phenoxyacid compounds may be prepared by a modification of Method 8151 (see Sec. 11.3 of this method) or other appropriate extraction technique. In general, the pH of an aqueous or a solid sample is adjusted to less than 2, and the sample is extracted with diethyl ether and concentrated, and the solvent exchanged to acetonitrile. Aqueous samples may also be extracted using solid-phase extraction after a pH adjustment, as described in Method 3535.
2.2.2 For carbamates, both aqueous and solid samples may be extracted with methylene chloride using a 3500 series method or other appropriate extraction technique, concentrated (preferably using a rotary evaporator equipped with an adapter) and the solvent is exchanged to methanol.
2.2.3 Samples for analysis of the other target analytes may be prepared by appropriate extraction techniques. In general, water samples may be extracted at a neutral pH with methylene chloride, using a 3500 series method or other appropriate extraction technique. Solid samples may be extracted with a mixture of methylene chloride/acetone (1:1), using a 3500 series method or other appropriate extraction technique. Extracts may need concentration and solvent exchange prior to analysis.
2.2.4 A micro-extraction technique for the extraction of tris (2,3-dibromopropyl) phosphate (abbreviated in this method as tris-BP) from aqueous and non-aqueous
matrices is included in this method (see Sec. 11.2). See the safety warning in Sec. 5.2 regarding this compound.
2.3 An optional mass spectrometry/mass spectrometry (MS/MS) confirmation procedure is provided in this method (see Sec. 11.11). That procedure employs MS/MS collision activated dissociation (CAD) or wire-repeller CAD.
3.0 DEFINITIONS
Refer to Chapter One and the manufacturer's instructions for definitions that may be relevant to this procedure.
4.0 INTERFERENCES
4.1 Solvents, reagents, glassware, and other sample processing hardware may yield discrete artifacts or elevated baselines, or both, causing misinterpretation of chromatograms or spectra. All of these materials must be demonstrated to be free from interferences under the conditions of the analysis by analyzing method blanks. Specific selection of reagents and purification of solvents by distillation in all-glass systems may be necessary. Refer to each method to be used for specific guidance on quality control procedures and to Chapter Four for general guidance on the cleaning of glassware.
4.2 Also refer to Methods 3500, 3600, 8000 and 8151 for discussions of interferences. 4.3 The use of Florisil® column cleanup (Method 3620) has been demonstrated to yield recoveries less than 85% for some of the compounds in this method, and is therefore not
recommended for all compounds. Refer to Method 3620 for recoveries of organophosphorus compounds as a function of Florisil® fractions.
4.4 Compounds with high proton affinity may mask the MS response of some of the target analytes. Therefore, except when the thermospray MS/MS system is used for rapid screening of samples (see Sec. 11.11.1), an HPLC must be used to perform the
chromatographic separations necessary for quantitative analyses.
4.5 Analytical difficulties encountered with specific organophosphorus compounds, as applied in this method, may include, but are not limited to, the following:
4.5.1 Methyl parathion shows some minor degradation during analysis. 4.5.2 Naled can undergo debromination to form dichlorvos (DDVP). This reaction may occur during sample preparation and extraction, and the extent may depend on the nature of the sample matrix. The analyst should consider the potential for
debromination of Naled when this compound is to be determined.
4.5.3 Merphos often contains contamination from merphos oxide. Oxidation of merphos can occur during storage, and possibly upon introduction into the mass
spectrometer.
4.5.4 The water solubility of DDVP is 10 g/L at 20 EC, and as a result, recovery of this compound by solvent extraction from aqueous solutions is poor.
4.5.5 Trichloron can rearrange and undergo dehydrochlorination (loss of HCl) in all types of media, forming DDVP. When either of these compounds are to be determined, the analyst should be aware of the possibility of this rearrangement in order to prevent misidentifications.
4.6 The chlorinated phenoxy acid compounds, being strong organic acids, react readily with alkaline substances and may be lost during analysis. Therefore, glassware and glass wool must be acid-rinsed, and sodium sulfate must be acidified with sulfuric acid prior to use, to avoid this possibility.
4.7 Due to the reactivity of the chlorinated herbicides, the standards must be prepared in acetonitrile. Methylation will occur slowly, if prepared in methanol.
4.8 Benomyl quickly degrades to carbendazim in the environment (Reference 7). 4.9 Interferants co-extracted from the sample will vary considerably from source to source. Retention times of target analytes must be verified by using reference standards.
4.10 The optional use of HPLC/MS/MS aids in the confirmation of specific analytes. These methods are less subject to chemical noise than other mass spectrometric methods.
4.11 It is unlikely that all 45 of the carbamates and related compounds that are listed in Sec. 1.1 can be determined in a single analysis. Based on a single-laboratory evaluation of 31 of the analytes (see Reference 11), the following analytes are known to co-elute under the HPLC conditions described in this method.
Detector Coeluting analytes
MS benomyl carbendazim
pebulate vernolate
physostigmine physostigmine salicylate
UV/Vis at 254 nm benomyl carbendazim
carbaryl carbofuran phenol
oxamyl formetanate hydrochloride
methiocarb/mexacarbate/promecarb/barban
UV/Vis at 280 nm benomyl carbendazim
barban mexacarbate
formetanate hydrochloride physostigmine salicylate
carbaryl carbofuran phenol
bendiocarb/propoxur/carbofuran
The differences between the groups of coeluting compounds for the two UV wavelengths are due to the fact that some of the coeluting compounds respond at one wavelength but do not respond at the other. The differences between the MS detector and the UV detectors is the result of the ability to resolve the mass spectra of some of the co-eluting compound, but not their UV responses.
5.0 SAFETY
5.1 This method does not address all safety issues associated with its use. The laboratory is responsible for maintaining a safe work environment and a current awareness file of OSHA regulations regarding the safe handling of the chemicals listed in this method. A reference file of material safety data sheets (MSDSs) should be available to all personnel involved in these analyses.
5.2 Tris-BP has been classified as a carcinogen. Purified standard material and stock standard solutions should be handled in a hood. Also, see Sec 11.2 for a description of micro-extraction procedures that may be applied to samples containing tris-BP. These procedures have been provided because the micro-extraction procedures should minimize the exposure of the analyst to samples or standards containing this compound, relative to the extraction of larger sample weights or volumes.
6.0 EQUIPMENT AND SUPPLIES
The mention of trade names or commercial products in this manual is for illustrative purposes only, and does not constitute an EPA endorsement or exclusive recommendation for use. The products and instrument settings cited in SW-846 methods represent those products and settings used during method development or subsequently evaluated by the Agency. Glassware, reagents, supplies, equipment, and settings other than those listed in this manual may be employed provided that method performance appropriate for the intended application has been demonstrated and documented.
6.1 HPLC/MS
The following apparatus and materials are necessary for the use of the HPLC/MS portions of this method.
6.1.1 High-performance liquid chromatograph (HPLC) -- An analytical system equipped with programmable solvent delivery system and all necessary accessories, including injection loop (with a minimum 10-µL loop volume), analytical columns, purging gases, etc. At a minimum, the solvent delivery system must be capable of delivering a binary solvent system. The chromatographic system must be capable of being interfaced with a mass spectrometer (MS).
6.1.2 HPLC post-column addition pump -- If post-column addition of reagents is employed, a pump is needed. Ideally, this pump should be a syringe pump, and does not have to be capable of solvent programming. It is also possible to add the ionization reagents to the solvents and not perform post-column addition (see Sec. 11.6.1).
6.1.3 HPLC/MS interface
6.1.3.1 Interface -- Thermospray ionization interface and source that will give acceptable calibration response for each analyte of interest at the
concentration required. The source must be capable of generating both positive and negative ions, and have a discharge electrode or filament. (See Figure 1.)
6.1.3.2 Micromixer -- 10-µL, connects HPLC column system with HPLC post-column addition solvent system, if post-column addition is used. 6.1.4 Mass spectrometer system
6.1.4.1 A single quadrupole mass spectrometer capable of scanning from 1 to 1000 amu. The spectrometer must also be capable of scanning from 150 to 450 amu in 1.5 sec. or less, using 70 volts (nominal) electron energy in the positive or negative electron impact modes. In addition, the mass spectrometer must be capable of producing a calibrated mass spectrum for PEG 400, 600, or 810 (see Sec. 7.15) or other compounds used for mass calibration.
6.1.4.2 Optional triple quadrupole mass spectrometer -- capable of generating daughter ion spectra with a collision gas in the second quadrupole and operation in the single quadrupole mode.
6.1.5 Data system -- A computer system that allows the continuous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program must be connected to the mass spectrometer. The computer must have software that allows any MS data file to be searched for ions of a specified mass, and such ion abundances to be plotted versus time or scan number. This type of plot is defined as an extracted ion current profile (EICP). Software must also be available that allows integration of the abundances in any EICP between specified time or scan-number limits. There must be computer software available to operate the specific modes of the mass spectrometer.
6.2 HPLC equipped with UV detector
An analytical system equipped with a solvent-programmable pumping system for at least a binary solvent system, and all necessary accessories including syringes, 10-µL injection loop, analytical columns, purging gases, etc. An automatic injector is optional, but is useful for multiple samples. The columns listed in Sec. 6.3 are also used with this system.
At a minimum, the UV detector must be capable of operating at the two wavelengths described in this procedure, e.g., 254 and 280 nm. Either conventional (photomultiplier tube) detectors or photodiode array detectors may be employed. If the UV detector is to be used in tandem with the thermospray interface, then the detector cell must be capable of withstanding high pressures (up to 6000 psi). However, the UV detector may be attached to an HPLC independent of the HPLC/MS and, in that case, standard HPLC pressures are acceptable.
6.3 HPLC columns
A guard column and an analytical column are necessary.
The columns listed in this section were those used in developing the method. The listing of these columns in this method is not intended to exclude the use of other columns that are available or that may be developed. Laboratories may use these columns or other columns, of other dimensions and/or packed with different stationary phases, provided that the laboratories document method performance data (e.g., chromatographic resolution, analyte breakdown, and sensitivity) that provide analytical performance that is appropriate for the intended application.
6.3.1 Guard column -- C18 reversed-phase guard column, 10 mm x 2.6 mm ID, 0.5-µm frit, or equivalent. The guard column should utilize the same or a similar stationary phase as the analytical column.
6.3.2 Analytical column -- C18 reversed-phase column, 100 mm x 2 mm ID, 5-µm particle size of ODS-Hypersil; or C8 reversed phase column, 100 mm x 2 mm ID, 3-µm particle size of MOS2-Hypersil, or equivalent
6.4 Purification equipment for azo dye standards 6.4.1 Soxhlet extraction apparatus
6.4.2 Extraction thimbles -- single thickness, 43 x 123 mm
6.4.3 Filter paper, 9.0 cm (Whatman qualitative No. 1 or equivalent) 6.4.4 Silica gel column -- 3 in. x 8 in., packed with silica gel (Type 60, EM reagent 70/230 mesh or equivalent)
6.5 Extraction equipment for chlorinated phenoxyacid compounds
6.5.1 Erlenmeyer flasks -- 500-mL wide-mouth glass, 500-mL glass with 24/40 ground-glass joint, 1000-mL glass
6.5.2 Separatory funnel -- 2000-mL 6.5.3 Graduated cylinder -- 1000-mL 6.5.4 Funnel -- 75-mm diameter
6.5.5 Wrist shaker -- Burrell Model 75 or equivalent 6.5.6 pH meter
6.6 Kuderna-Danish (K-D) apparatus (optional)
6.6.1 Concentrator tube -- 10-mL graduated. A ground-glass stopper is used to prevent evaporation of extracts.
6.6.2 Evaporation flask -- 500-mL. Attach to concentrator tube with springs, clamps, or equivalent.
6.6.3 Two-ball micro-Snyder column 6.6.4 Springs -- ½-in.
6.6.5 Solvent vapor recovery system (Kontes K-545000-1006 or K-547300-0000, Ace Glass 6614-30, or equivalent).
NOTE: This glassware is recommended for the purpose of solvent recovery during the concentration procedures requiring the use of Kuderna-Danish evaporative concentrators. Incorporation of this apparatus may be required by Federal, State or local municipality regulations that govern air emissions of volatile
organics. EPA recommends the incorporation of this type of reclamation system as a method to implement an emissions reduction program. Solvent recovery is a means to conform with waste minimization and pollution prevention initiatives. 6.7 Disposable serological pipets
6.8 Collection tube -- 15-mL conical, graduated
6.9 Vials -- 5-mL conical, glass, fitted with polytetrafluoroethylene (PTFE)-lined screw-caps or crimp tops
6.10 Silanized glass wool
6.11 Microsyringes -- 100-µL, 50-µL, 10-µL (Hamilton 701 N or equivalent), and 50-µL blunted (Hamilton 705SNR or equivalent)
6.12 Rotary evaporator -- Equipped with 1000-mL receiving flask
6.13 Balances -- Analytical capable of weighing to 0.0001 g, and top-loading capable of weighing to 0.01 g
6.14 Volumetric flasks, Class A -- 10-mL to 1000-mL 6.15 Graduated cylinder -- 100-mL
6.16 Separatory funnel -- 250-mL
6.17 Separatory funnel -- 2-L, fitted with PTFE stopcock
6.19 Nitrogen evaporation apparatus -- N-Evap Analytical Evaporator Model 111, Organomation Association Inc., Northborough, MA, or equivalent
7.0 REAGENTS AND STANDARDS
7.1 Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, it is intended that all reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening the accuracy of the determination. Reagents should be stored in glass to prevent the leaching of contaminants from plastic containers.
7.2 Organic-free reagent water. All references to water in this method refer to organic-free reagent water, as defined in Chapter One.
7.3 Sodium sulfate (granular, anhydrous), Na2SO4. Purify by heating at 400 EC for 4 hr in a shallow tray, or by precleaning with methylene chloride.
7.4 Ammonium acetate, NH4OOCCH3, solution (0.1 M). Filter through a 0.45-µm membrane filter (Millipore HA or equivalent).
7.5 Acetic acid, CH3CO2H (1% v/v and 0.1N)
7.6 Sulfuric acid solution -- 1:1, v/v. Prepare by slowly adding 50 mL of H2SO4 (sp. gr. 1.84) to 50 mL of water.
7.7 Argon gas, 99+% pure 7.8 Extraction solvents
Samples should be extracted using a solvent system that gives optimum, reproducible recovery of the analytes of interest from the sample matrix, at the concentrations of interest. The choice of extraction solvent will depend on the analytes of interest and no single solvent is universally applicable to all analyte groups. Whatever solvent system is employed, including those specifically listed in this method, the analyst must demonstrate adequate performance for the analytes of interest, at the levels of interest. At a minimum, such a demonstration will encompass the initial demonstration of proficiency described in Method 3500, using a clean reference matrix. Method 8000 describes procedures that may be used to develop performance criteria for such demonstrations as well as for matrix spike and laboratory control sample
results.
Unless otherwise noted, all solvents must be HPLC-grade or equivalent. Solvents may be degassed prior to use.
7.8.1 Methylene chloride, CH2Cl2 7.8.2 Toluene, C6H5CH3
7.8.3 Acetone, CH3COCH3
7.8.4 Diethyl ether, C2H5OC2H5 -- Must be free of peroxides as indicated by test strips (EM Quant, or equivalent). Procedures for removal of peroxides are provided with
the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.
7.8.5 Methanol, CH3OH - HPLC-grade or equivalent 7.8.6 Acetonitrile, CH3CN - HPLC-grade or equivalent 7.8.7 Ethyl acetate, CH3CO2C2H5
7.9 Standard materials -- Pure standard materials or certified solutions of each analyte targeted for analysis. Disperse azo dyes must be purified before use according to Sec. 7.10. WARNING: Tris-BP has been classified as a carcinogen. Purified standard material and stock
standard solutions should be handled in a hood. 7.10 Disperse azo dye purification
Two procedures are involved. The first step is the Soxhlet extraction of the dye for 24 hr with toluene and evaporation of the liquid extract to dryness, using a rotary evaporator. The solid is then recrystallized from toluene, and dried in an oven at approximately 100 EC. If this step does not give the necessary purity, column chromatography should be employed. Load the solid onto a silica gel column (Sec. 6.4.4), and elute with diethyl ether. Separate the impurities using chromatography, and collect the major dye fraction.
7.11 Standard solutions
The following sections describe the preparation of stock, intermediate, and working standards for the compounds of interest. This discussion is provided as an example, and other approaches and concentrations of the target compounds may be used, as appropriate for the intended application. See Method 8000 for additional information on the preparation of calibration standards.
7.12 Stock standard solutions
Standards may be prepared from pure standard materials or may be purchased as
certified solutions. Commercially-prepared stock standards may be used if they are certified by the manufacturer and verified against a standard made from pure material.
7.12.1 Prepare stock standard solutions by accurately weighing 0.0100 g of pure material. Dissolve the material in methanol or other suitable solvent (e.g., prepare tris-BP in ethyl acetate), and dilute to known volume in a volumetric flask.
NOTE: Due to the reactivity of the chlorinated herbicides, the standards must be prepared in acetonitrile. Methylation will occur if standards are prepared in methanol.
If compound purity is certified at 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially-prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.
7.12.2 Transfer the stock standard solutions into glass vials fitted with PTFE-lined screw-caps or crimp-tops. Store at #6 EC and protect from light. Stock standard
solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards.
7.13 Calibration standards
A minimum of five different concentrations for each parameter of interest should be prepared through dilution of the stock standard solutions with methanol (or other suitable solvent). At least one of the calibration standards should correspond to a sample concentration at or below that necessary to meet the performance objectives of the project. The remaining concentrations should correspond to the expected range of concentrations found in real samples, or should define the working range of the HPLC-UV or HPLC-TS/MS system (see Method 8000). Calibration standards should be replaced after one or two months, or sooner if comparison with check standards indicates a problem. See Method 8000 for additional information regarding the preparation of calibration standards.
7.14 Surrogate standards
The analyst should monitor the performance of the extraction, cleanup (when used), and analytical system, along with the effectiveness of the method in dealing with each sample matrix, by spiking each sample, standard, and blank with one or two surrogates (e.g.,
organophosphorus or chlorinated phenoxyacid compounds not expected to be present in the sample).
7.15 HPLC/MS tuning standard
Polyethylene glycol 400 (PEG 400), PEG 600, or PEG 810 are recommended as tuning standards. However, analysts may use other tuning standards as recommended by the instrument manufacturer or other documented source. If one of the PEG solutions is used, dilute to 10 percent (v/v) in methanol. Which PEG is used will depend upon analyte molecular weight range of the analytes of interest. For molecular weights less than 500, use PEG 400, and for molecular weights greater than 500, use PEG 600 or PEG 810.
7.16 Internal standards
When the internal standard calibration option is used for HPLC/MS analyses, it is recommended that analysts use stable isotopically-labeled compounds of the same chemical class when they are available (e.g., 13C
6-carbofuran may be used as an internal standard in the analysis of carbamates).
7.17 Matrix spiking standards
Consult Method 3500 for information on matrix spiking solutions. Prepare a solution containing the analytes of interest in a suitable solvent.
NOTE: The form of the compounds used for spiking should be identical to the form of the target analytes. For the phenoxyacid herbicides in particular, use the acid form of the acid analytes, not the ester form or an ether, because use of these other forms will not represent the performance of the overall extraction, cleanup, and determinative
methods relative to the target analytes. Conversely, when the ester forms are of the analytes of interest, e.g., 2,4-D, butoxyethanol ester, use the ester form of the analyte for preparing matrix spiking solutions.
8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE See the introductory material to Chapter Four, "Organic Analytes."
9.0 QUALITY CONTROL
9.1 Refer to Chapter One for guidance on quality assurance (QA) and quality control (QC) protocols. When inconsistencies exist between QC guidelines, method-specific QC criteria take precedence over both technique-specific criteria and those criteria given in Chapter One, and technique-specific QC criteria take precedence over the criteria in Chapter One. Any effort involving the collection of analytical data should include development of a structured and systematic planning document, such as a Quality Assurance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which translates project objectives and specifications into directions for those that will implement the project and assess the results. Each laboratory should maintain a formal quality assurance program. The laboratory should also maintain records to document the quality of the data generated. All data sheets and quality control data should be maintained for reference or inspection.
9.2 Refer to Method 8000 for specific determinative method QC procedures. Refer to Method 3500 for QC procedures to ensure the proper operation of the various sample
preparation techniques. If an extract cleanup procedure is performed, refer to Method 3600 for the appropriate QC procedures. Any more specific QC procedures provided in this method will supersede those noted in Methods 8000, 3500, or 3600.
9.3 The quality control procedures necessary to evaluate the HPLC system operation are found in Method 8000 and include evaluation of retention time windows, calibration
verification and chromatographic analysis of samples. When the mass spectrometer is used as the detector, it must be tuned at the beginning of each 12-hour analytical shift. Check the performance of the entire analytical system daily using data gathered from analyses of blanks, standards, and replicate samples. If any of the chromatographic QC limits are not met, the analyst should examine the LC system for:
• Leaks,
• Proper pressure delivery,
• A dirty guard column; may need replacing or repacking, and • Possible partial thermospray plugging.
Checking any of the above items will necessitate shutting down the HPLC/TS system, making repairs and/or replacements, and then restarting the analyses. A tuning standard and a calibration verification standard should be reanalyzed before any sample analyses, as described in Sec. 11.8.3.
9.4 Initial demonstration of proficiency
Each laboratory must demonstrate initial proficiency with each sample preparation and determinative method combination it utilizes by generating data of acceptable accuracy and precision for target analytes in a clean matrix. If an autosampler is used to perform sample dilutions, before using the autosampler to dilute samples, the laboratory should satisfy itself that those dilutions are of equivalent or better accuracy than is achieved by an experienced analyst performing manual dilutions. The laboratory must also repeat the demonstration of proficiency whenever new staff members are trained or significant changes in instrumentation are made. See Method 8000 for information on how to accomplish a demonstration of proficiency.
9.5 Initially, before processing any samples, the analyst should demonstrate that all parts of the equipment in contact with the sample and reagents are interference-free. This is accomplished through the analysis of a method blank. As a continuing check, each time samples are extracted, cleaned up, and analyzed, and when there is a change in reagents, a method blank should be prepared and analyzed for the compounds of interest as a safeguard against chronic laboratory contamination. The blanks should be carried through all stages of sample preparation and analysis. If a peak is observed within the retention time window of any analyte that would prevent the determination of that analyte, determine the source and eliminate it, if possible, before processing samples. The blanks should be carried through all stages of sample preparation and analysis. When new reagents or chemicals are received, the laboratory should monitor the preparation and/or analysis blanks associated with samples for any signs of contamination. It is not necessary to test every new batch of reagents or chemicals prior to sample preparation if the source shows no prior problems. However, if reagents are changed during a preparation batch, separate blanks need to be prepared for each set of reagents.
9.6 Sample quality control for preparation and analysis
The laboratory must also have procedures for documenting the effect of the matrix on method performance (precision, accuracy, method sensitivity). At a minimum, this should include the analysis of QC samples including a method blank, a matrix spike, a duplicate, and a laboratory control sample (LCS) in each analytical batch, the addition of surrogates to each field sample and QC sample when surrogates are used. Any method blanks, matrix spike samples, and replicate samples should be subjected to the same analytical procedures (Sec. 11.0) as those used on actual samples.
9.6.1 Documenting the effect of the matrix should include the analysis of at least one matrix spike and one duplicate unspiked sample or one matrix spike/matrix spike duplicate pair. The decision on whether to prepare and analyze duplicate samples or a matrix spike/matrix spike duplicate must be based on a knowledge of the samples in the sample batch. If samples are expected to contain target analytes, then laboratories may use a matrix spike and a duplicate analysis of an unspiked field sample. If samples are not expected to contain target analytes, the laboratories should use a matrix spike and matrix spike duplicate pair. Consult Method 8000 for information on developing
acceptance criteria for the MS/MSD.
9.6.2 A laboratory control sample (LCS) should be included with each analytical batch. The LCS consists of an aliquot of a clean (control) matrix similar to the sample matrix and of the same weight or volume. The LCS is spiked with the same analytes at the same concentrations as the matrix spike, when appropriate. When the results of the matrix spike analysis indicate a potential problem due to the sample matrix itself, the LCS results are used to verify that the laboratory can perform the analysis in a clean matrix. Consult Method 8000 for information on developing acceptance criteria for the LCS. 9.6.3 Also see Method 8000 for the details on carrying out sample quality control procedures for preparation and analysis. In-house method performance criteria for evaluating method performance should be developed using the guidance found in Method 8000.
9.7 Surrogate recoveries
If surrogates are used, the laboratory should evaluate surrogate recovery data from individual samples versus the surrogate control limits developed by the laboratory. See Method 8000 for information on evaluating surrogate data and developing and updating surrogate limits. Procedures for evaluating the recoveries of multiple surrogates and the associated corrective actions should be defined in an approved project plan.
9.8 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.
10.0 CALIBRATION AND STANDARDIZATION
See Sec 11.8 for information on calibration and standardization. 11.0 PROCEDURE
11.1 Sample preparation
Prior to analysis, samples must be extracted using an appropriate 3500 series method, the specific procedures described in this method, or another extraction procedure appropriate for the sample matrix. The analyst should choose among the possible extraction procedures based on the nature of the samples and the sensitivity required for the intended application.
Whatever extraction procedure is employed, including those specifically listed in this method, the analyst must demonstrate adequate performance for the analytes of interest, at the levels of interest. At a minimum, such a demonstration will encompass the initial demonstration of proficiency described in Method 3500, using a clean reference matrix. Method 8000
describes procedures that may be used to develop performance criteria for such demonstrations as well as for matrix spike and laboratory control sample results.
11.1.1 Samples for the analysis of disperse azo dyes and organophosphorus compounds must be prepared by an appropriate 3500 series method, or other appropriate extraction procedure, prior to HPLC/MS analysis.
11.1.2 Samples for the analysis of tris-BP must be prepared according to Sec. 11.2, prior to HPLC/MS analysis, unless the analyst can demonstrate appropriate performance for the intended application using other techniques.
11.1.3 Samples for the analysis of chlorinated phenoxyacid compounds and their esters should be prepared according to Sec. 11.3, or other appropriate technique, prior to HPLC/MS analysis. TCLP leachates to be analyzed for the phenoxyacid herbicides may also be prepared using solid-phase extraction (SPE), as described in Method 3535. 11.2 Microextraction of tris-BP
The following sections describe micro-extraction procedures that may be applied to samples containing tris-BP. These procedures have been provided because tris-BP is a known carcinogen, and the micro-extraction procedures should reduce the exposure of the analyst to samples or standards containing this compound over the extraction of larger sample weights or volumes.
11.2.1 Solid samples
11.2.1.1 Weigh a 1-g portion of the sample into a tared beaker. If the sample appears moist, add an equivalent amount of anhydrous sodium sulfate and mix well. Add 100 µL of tris-BP (approximate concentration 1000 mg/L) to the
% dry weight ' g of dry sample g of sample ×100
sample selected for spiking; the amount added should result in a final concentration of 100 ng/µL in the 1-mL extract.
11.2.1.2 Remove the glass wool plug from a disposable serological pipet. Insert a 1-cm plug of clean silane-treated glass wool to the bottom (narrow end) of the pipet. Pack 2 cm of anhydrous sodium sulfate on top of the glass wool. Wash the pipet and its contents with 3 - 5 mL of methanol.
11.2.1.3 Pack the sample into the pipet. If the packing material has dried, begin by wetting it with a few mL of methanol, then pack the sample into the pipet.
11.2.1.4 Extract the sample with 3 mL of methanol followed by 4 mL of 50% (v/v) methanol/methylene chloride. Rinse the sample beaker with each volume of extraction solvent prior to adding it to the pipet containing the sample. Collect the extract in a 15-mL graduated glass tube.
11.2.1.5 Concentrate the extract to 1 mL using the nitrogen evaporation technique (see Sec. 11.5). Record the volume. (It may not be possible to
evaporate some sludge samples to a 1-mL final volume.)
11.2.1.6 Determination of percent dry weight -- When sample results are to be calculated on a dry weight basis, a separate portion of sample should be weighed out at the same time as the portion used for analytical determination. CAUTION: The drying oven should be contained in a hood or vented.
Significant laboratory contamination may result from drying a heavily contaminated hazardous waste sample.
Immediately after weighing the sample for extraction, weigh an additional 5 - 10 g of the sample into a tared crucible. Dry this aliquot overnight at 105 EC. Allow it to cool in a desiccator before weighing. Calculate the % dry weight as follows:
NOTE: This dry weight determination also applies to the use of other extraction techniques for solids (e.g., a 3500 series method), when results are to be calculated on a dry weight basis.
This oven-dried aliquot is not used for the extraction and should be disposed of appropriately once the dry weight is determined.
11.2.2 Aqueous samples
11.2.2.1 Using a 100-mL graduated cylinder, measure 100 mL of sample and transfer it to a 250-mL separatory funnel. Add 200 µL of tris-BP (approximate concentration 1000 mg/L) to the sample selected for spiking; the amount added should result in a final concentration of 200 ng/µL in the 1-mL extract.
11.2.2.2 Add 10 mL of methylene chloride to the separatory funnel. Seal and shake the separatory funnel three times, approximately 30 sec each time, with periodic venting to release excess pressure.
WARNING: Methylene chloride creates excessive pressure rapidly; therefore, initial venting should be done immediately after the separatory funnel has been sealed and shaken once. Methylene chloride is a
suspected carcinogen, use necessary safety precautions.
11.2.2.3 Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between the layers is more than one-third the size of the solvent layer, the analyst must employ mechanical techniques to complete phase separation. See Method 3510.
11.2.2.4 Collect the extract in a 15-mL graduated glass tube. Concentrate the extract to 1 mL, using nitrogen evaporation (see Sec. 11.5). 11.3 Extraction for chlorinated phenoxyacid compounds
Preparation of soil, sediment, and other solid samples should follow the procedures outlined in Method 8151, or other appropriate technique, except that no hydrolysis or
esterification generally is necessary. However, it is recommended that to determine all of the phenoxyacid moieties as the acid, hydrolysis to the acid form should be performed in order to simplify quantitation. Sec. 11.3.1 presents an outline of the procedure with the appropriate changes necessary for determination by this method. Sec. 11.3.2 describes the extraction procedure for aqueous samples. TCLP leachates may be extracted using solid-phase extraction, as described in Method 3535.
11.3.1 Extraction of solid samples
11.3.1.1 Add 50 g of soil/sediment sample to a 500-mL, wide-mouth Erlenmeyer flask. Add spiking solutions, if needed. Mix well and allow to stand for 15 min. Add 50 mL of organic-free reagent water and stir for 30 min. Determine the pH of the sample with a glass electrode and pH meter, while stirring. Adjust the pH to 2 with cold dilute (1:1) H2SO4 and monitor the pH for 15 min, with stirring. If necessary, add additional H2SO4 until the pH remains at 2. Other sample
weights and reagent volumes may be employed, provided that the laboratory can demonstrate acceptable sensitivity and performance for the intended application. 11.3.1.2 Add 20 mL of acetone to the flask, and mix the contents with the wrist shaker for 20 min. Add 80 mL of diethyl ether to the same flask, and shake again for 20 min. Decant the extract and measure the volume of solvent recovered.
11.3.1.3 Extract the sample twice more using 20 mL of acetone followed by 80 mL of diethyl ether. After addition of each solvent, the mixture should be shaken with the wrist shaker for 10 min and the acetone-ether extract decanted.
11.3.1.4 After the third extraction, the volume of extract recovered should be at least 75% of the volume of added solvent. If this is not the case, additional extractions may be necessary. Combine the extracts in a 2000-mL separatory funnel containing 250 mL of 5% acidified sodium sulfate. If an emulsion forms, slowly add 5 g of acidified sodium sulfate (anhydrous) until the
solvent-water mixture separates. A quantity of acidified sodium sulfate equal to the weight of the sample may be added, if necessary.
11.3.1.5 Check the pH of the extract. If it is not at or below pH 2, add more dilute H2SO4 until the extract is stabilized at the desired pH. Gently mix the contents of the separatory funnel for 1 min and allow the layers to separate. Collect the aqueous phase in a clean beaker, and collect the extract phase (top layer) in a 500-mL Erlenmeyer flask equipped with a ground-glass stopper. Place the aqueous phase back into the separatory funnel and re-extract using 25 mL of diethyl ether. Allow the layers to separate and discard the aqueous layer. Combine the ether extracts in the 500-mL Erlenmeyer flask.
11.3.1.6 Add 45 - 50 g of acidified anhydrous sodium sulfate to the combined ether extracts. Allow the extract to remain in contact with the sodium sulfate for approximately 2 hr.
CAUTION: The drying step is critical. Any moisture remaining in the ether will result in low recoveries. The amount of sodium sulfate used is adequate if some free flowing crystals are visible when swirling the flask. If all of the sodium sulfate solidifies in a cake, add a few
additional grams of acidified sodium sulfate and again test by swirling. The 2-hr drying time is a minimum; and the extracts may be held overnight in contact with the sodium sulfate.
11.3.1.7 Transfer the ether extract, through a funnel plugged with acid-washed glass wool, into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Use a glass rod to crush caked sodium sulfate during the transfer. Rinse the Erlenmeyer flask and column with 20 - 30 mL of diethyl ether to complete the quantitative transfer. Reduce the volume of the extract using the macro K-D technique (see Sec. 11.5).
11.3.2 Extraction of aqueous samples
11.3.2.1 Using a 1000-mL graduated cylinder, measure 1 liter (nominal) of sample, record the sample volume to the nearest 5 mL, and transfer it to a separatory funnel. If high concentrations are anticipated, a smaller volume may be used and then diluted with organic-free reagent water to 1 liter. Adjust the pH to 2 with dilute (1:1) H2SO4. Other sample weights and reagent volumes may be employed, provided that the laboratory can demonstrate acceptable sensitivity and performance for the intended application.
11.3.2.2 Add 150 mL of diethyl ether to the sample bottle, seal, and shake for 30 sec to rinse the walls. Transfer the solvent wash to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water layer for a minimum of 10 min. If the emulsion interface between the layers is more than one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, and may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Drain the aqueous phase into a 1000-mL Erlenmeyer flask.
11.3.2.3 Repeat the extraction two more times using 100 mL of diethyl ether each time. Combine the extracts in a 500-mL Erlenmeyer flask. (Rinse the
1000-mL flask with each additional aliquot of extraction solvent to make a quantitative transfer.)
11.3.2.4 Proceed to Sec. 11.5 for drying, K-D concentration, solvent exchange, and final volume adjustment.
11.4 Extraction of carbamates
Preparation of aqueous, soil, sediment, and other solid samples may be accomplished using an appropriate 3500 series method. The following sections provide general
considerations.
11.4.1 One-liter aqueous samples may be extracted with methylene chloride using an appropriate 3500 series method.
11.4.2 Forty-gram quantities of solid samples may be extracted with methylene chloride using an appropriate 3500 series method.
11.4.3 Concentration steps may be performed using a rotary evaporator or K-D, reducing the volume of the final extract to 5 - 10 mL.
11.4.4 Final concentration of the extract and exchanging the solvent to a 1-mL final volume of methanol may be accomplished using an adaptor on the rotary evaporator. If an adaptor is unavailable, the final concentration may be performed using nitrogen evaporation, in a fume hood.
11.4.5 Other sample weights and reagent volumes may be employed, provided that the laboratory can demonstrate acceptable sensitivity and performance for the intended application.
11.5 Extract concentration techniques
Two procedures are provided for the concentration of extracts: macro-concentration by Kuderna-Danish (K-D) and micro-concentration by nitrogen evaporation.
11.5.1 Macro-concentration by K-D
Add one or two clean boiling chips to the flask and attach a three-ball
macro-Snyder column. Attach the solvent vapor recovery glassware (condenser and collection device, Sec. 6.6.5) to the Snyder column of the K-D apparatus following manufacturer's instructions. Prewet the Snyder column by adding about 1 mL of diethyl ether to the top. Place the apparatus on a hot water bath (60 EC - 65 EC) so that the concentrator tube is partially immersed in the hot water and the entire lower rounded surface of the flask is bathed in vapor. Adjust the vertical position of the apparatus and the water temperature, as necessary, to complete the concentration in 15 - 20 min. At the proper rate of distillation the balls of the column will actively chatter, but the chambers will not flood. When the apparent volume of liquid reaches 5 mL, remove the K-D apparatus from the water bath and allow it to drain and cool for at least 10 min.
11.5.2 Solvent exchange
11.5.2.1 Transfer the concentrator tube to a nitrogen evaporation device. Add a total of 5 mL of the final solvent of choice (methanol or acetonitrile).
11.5.2.2 Reduce the extract volume according to Sec. 11.5.3 and adjust the final volume to 1 mL (or other volume necessary to achieve the required sensitivity).
11.5.3 Micro-concentration by nitrogen evaporation
11.5.3.1 Place the concentrator tube in a warm water bath
(approximately 35 EC) and evaporate the solvent volume to the designated level using a gentle stream of clean, dry nitrogen (filtered through a column of activated carbon).
CAUTION: Do not use plasticized tubing between the carbon trap and the sample.
11.5.3.2 The internal wall of the tube must be rinsed down several times with the final solvent during the operation. During evaporation, the solvent level in the tube should be positioned to prevent water from condensing into the sample (i.e., the solvent level should be below the level of the water bath). Under normal operating conditions, the extract should not be allowed to become dry. 11.5.4 Transfer the extract to a glass vial fitted with a PTFE-lined screw-cap or crimp-top and store refrigerated at #6 EC. Proceed with HPLC analysis.
11.6 HPLC chromatographic conditions
11.6.1 Suggested mobile phases and elution gradients for some groups of analytes are shown in Tables 1 and 2. Analysts should also consult the instrument manufacturer’s instructions. In the absence of specific suggestions, the following conditions may be a useful starting point:
Flow rate 0.8 mL/min
Post-column mobile phase 0.1 M ammonium acetate (use [1% methanol]/[0.1 M ammonium acetate for phenoxyacid compounds]) Post-column flow rate 0.4 mL/min
Optimize the instrumental conditions for resolution of the target analytes and sensitivity. Post-column addition of the MS ionization reagents may not be necessary in all instances, and these reagents may be added to the elution solvents, provided that adequate
performance can be demonstrated.
NOTE: Once established, the same operating conditions must be used for both calibrations and sample analyses.
11.6.2 If there is a chromatographic problem when analyzing disperse azo dyes, organophosphorus compounds, or tris-BP, apply a 2% constant flow of methylene
chloride, as needed. Methylene chloride/aqueous methanol solutions must be used with caution as HPLC eluants. Acetic acid (1%), another mobile phase modifier, can be used with compounds with acid functional groups.
11.6.3 A total flow rate of 1.0 - 1.5 mL/min may be necessary to maintain thermospray ionization, however, consult the instrument manufacturer's instructions and adjust the flow rate as needed.
11.7 Suggested thermospray/MS operating conditions
Prior to analysis of samples, the analyst should evaluate the relative sensitivity of the target compounds to each ionization mode to determine which may provide better sensitivity during analyses. This evaluation may be based on the structures of the analytes or on analyses conducted. Some groups of target compounds will have much better sensitivity using either positive or negative ionization (e.g., carbamates are generally more sensitive to the positive ionization mode and phenoxyacids are generally more sensitive to the negative ionization mode). When all the analytes of interest for a given application respond adequately in a given ionization mode, a single analysis using that mode may be employed.
11.7.1 Positive ionization mode conditions
Discharge electrode Off
Filament On or off (optional, analyte-dependent)
Mass range 150 450 amu (analytedependent, expect 1 -18 amu higher than the molecular weight of the compound).
Scan time 1.50 sec/scan
Optional repeller wire or plate 170 - 250 v (sensitivity optimized). See Figure 2 for schematic of source equipped with wire repeller.
11.7.2 Negative ionization mode conditions
Discharge electrode On
Filament Off
Mass range 135 - 450 amu
Scan time 1.50 sec/scan
11.7.3 Thermospray temperatures
Vaporizer control 110 - 130 EC
Vaporizer tip 200 - 215 EC
Jet 210 - 220 EC
Source block 230 - 265 EC. (Some compounds may degrade in the source block at higher temperatures. The operator should use knowledge of the chemical properties of the analytes of interest to estimate proper source temperature.)
11.7.4 Sample injection volume
An injection volume of 20 - 100 µL is normally used. The injection loop must be overfilled by at least a factor of two (e.g., 20 µL of the sample is used to overfill a 10-µL injection loop) when manual injections are performed. If solids are present in the extract, allow them to settle or centrifuge the extract and withdraw the injection volume from the clear layer.
11.8 Calibration
11.8.1 Tuning the mass spectrometer system
When an MS detector is employed, the detector must be tuned at the beginning of each 12-hr analytical shift using the procedures outlined in Secs. 11.8.1.1 and 11.8.1.2, to ensure correct mass assignments, sensitivity, and resolution. Tuning may be
accomplished using polyethylene glycol (PEG) 400, 600, or 810 (see Sec. 7.15) which have average molecular weights of 400, 600, and 810, respectively. Analysts may use other tuning standards as recommended by the instrument manufacturer or other
documented source. If PEGs are used, a mixture of these PEGs can be made such that it will approximate the expected working mass range for the analyses. Use PEG 400 for analysis of chlorinated phenoxyacid compounds.
The PEG is introduced via the thermospray interface, circumventing the HPLC. For triple quadrupole systems, both quadrupole 1 and quadrupole 3 are tuned.
11.8.1.1 The mass calibration parameters are as follows:
PEG 400 and 600 PEG 810
Mass range 15 to 765 amu Mass range 15 to 900 amu Scan time 0.5 to 5.0 sec/scan Scan time 0.5 to 5.0 sec/scan
Approximately 100 scans should be acquired, with 2 or 3 injections made. The scan with the best fit to the accurate mass table (see Tables 7 and 8) should be used as the calibration table. If a mass calibrant other than PEG is used, then the mass range should extend approximately 15 - 20 amu higher than the highest mass used for calibration. A scan time should be chosen which will give at least 6 scans across the calibrant peak.
11.8.1.2 The low mass range from 15 to 100 amu is covered by the ions from the ammonium acetate buffer used in the thermospray process.
NH4+ 18 amu NH4 + C H 20 36 amu CH3OH C NH4 + 50 amu (methanol) CH3 CN C NH4+ 59 amu (acetonitrile) CH3OOH C NH 4 +
78 amu (acetic acid)
The appearance of m/z 50 or 59 depends upon the use of methanol or acetonitrile as the organic modifier. The higher mass range is covered by the ammonium ion adducts of the various ethylene glycols (e.g., H(OCH2CH2)nOH where n=4, gives the H(OCH2CH2)4OHCNH4+ ion at m/z 212).
11.8.2 Liquid chromatographic system
11.8.2.1 Choose the proper ionization conditions for the MS detector, as outlined in Sec. 11.7. When UV detection is employed in conjunction with the MS detector, establish appropriate operating conditions for the UV detector.
11.8.2.2 Prepare at least five calibration standards (see Sec. 7.11 and Method 8000). Inject each calibration standard onto the HPLC, using the
chromatographic conditions outlined in Table 1. Refer to Method 8000 for guidance on external and internal calibration options and calibration acceptance criteria. In most cases the (M+H)+ and (M +NH
4)
+ adduct ions are the only ions of significant abundance. For example, Table 9 lists example retention times and the major ions (>5%) present in the positive ionization thermospray single quadrupole spectra of the organophosphorus compounds. (The retention times listed in Table 9 are provided for illustrative purposes only. Each laboratory must determine retention times and retention time widows for their specific application of the method.)
11.8.2.3 Selective ion monitoring (SIM) may be used in situations requiring detection limits below the normal range of full spectra analysis. However, SIM may provide a lesser degree of confidence in the compound identification unless multiple ions are monitored for each compound.
11.8.2.4 The use of selective reaction monitoring (SRM) is also acceptable when using triple-quad MS/MS and enhanced sensitivity is needed.
11.8.2.5 If UV detection is being used, integrate the area under the full chromatographic peak for each concentration. Quantitation by HPLC-UV may be preferred if it is known that sample interference and/or analyte coelution are not a problem, or when response of the MS detector is not sufficiently stable for
quantitative analyses. In these instances, the MS response may be used for positive qualitative identification of the analytes while the UV response is used for quantitation.
11. 8.2.6 The retention time of the chromatographic peak is an
important parameter in analyte identification. Therefore, the relative retention time of the analyte (versus the internal standard) should be in the range of 0.9 to 1.1. 11.8.3 Calibration verification
At the beginning of each 12-hr analytical shift, the response of the instrument system must be verified by the analysis of the MS tuning (if the MS detector is used) and by the analysis of a calibration verification standard at the approximate mid-point of the initial calibration range. Consult Sec. 11.8.1 and Method 8000 for information on performing these demonstrations and the acceptance criteria that should be employed. 11.9 Sample analysis
Once the LC system has been calibrated as outlined in Sec. 11.8, sample analysis may begin, employing either the MS or UV detector, or both. Depending on the sensitivity necessary for a given project, analyses may be conducted using the MS detector in either the positive or negative ionization modes. The positive ionization mode generally provides greater sensitivity, and may be more appropriate for samples containing very low concentrations of the analytes of
interest. However, analysts are advised that some compounds may be detectable in only the negative ionization mode.
11.9.1 An instrument blank (methanol) should be analyzed after the standards, in order to demonstrate that the system is free from contamination.
11.9.2 If performing manual injections, take an appropriate aliquot of the sample as per Sec. 11.7.4. Start the HPLC gradient elution, load and inject the sample aliquot, and start the mass spectrometer data system.
11.9.3 When using an autoinjector, ensure that it is set up according to the manufacturer's instructions and that all samples and standards are loaded in the proper order. Start the autoinjector, the HPLC gradient elution, and the MS data system.
11.9.4 The concentration of the analyte is determined by using the initial
calibration data (see Method 8000) from either the MS or UV detector response. Samples whose concentrations exceed the calibration range must be diluted to fall within the range. 11.9.5 When using MS or MS/MS, and when it is appropriate for the compounds of interest and the project objectives, determinations in both positive and negative
ionization analyses may be done on each sample extract.
11.9.6 If only the UV detector is used, then consult Method 8000 for information on the confirmation of analyte identifications.
11.10 Calculations
11.10.1 Using the external or internal standard calibration procedure (Method 8000), determine the identity and quantity of each component peak in the sample
reconstructed ion chromatogram which corresponds to the compounds used for calibration processes. See Method 8000 for calculations.
11.10.2 The retention time of the chromatographic peak is an important parameter in analyte identification. However, because matrix interferences can change
chromatographic column conditions, and thereby alter retention times, the absolute retention times are not as significant in analyte identification as relative retention times (when using internal standards). In addition, the mass spectral patterns are important criteria for analyte identification.
11.10.3 In instances when the TS/MS response exhibits higher variability, the MS response may be used to identify the analytes of interest while the quantitative results are derived from the response of the UV detector.
11.11 Optional MS/MS confirmation
With respect to this method, MS/MS is defined as daughter ion collision-activated dissociation acquisition, with quadrupole 1 set on one mass (parent ion), quadrupole 2 pressurized with argon and with a higher offset voltage than normal, and quadrupole 3 set to scan the desired mass range.
11.11.1 Since the thermospray process often generates only one or two ions per compound, the use of MS/MS is a more specific mode of operation yielding molecular structural information. In this mode, samples can be rapidly screened through direct
injection of the sample into the thermospray (e.g., without using the HPLC to separate the sample components).
11.11.2 When using MS/MS, quadrupole 1 should be set to the protonated molecule or ammoniated adduct of the analyte of interest. Quadrupole 3 should be set to scan from 30 amu to just above the mass region of the protonated molecule.
11.11.3 The collision gas pressure (Ar) should be set at about 1.0 mTorr and the collision energy at 20 eV. If these parameters fail to give considerable fragmentation, the settings may be increased to create more and stronger collisions.
11.11.4 For analytical determinations, the base peak of the collision spectrum must be used as the quantitation ion. For extra specificity, a second ion should be chosen as a backup quantitation ion.
11.11.5 Perform an initial calibration, as outlined in Sec. 11.8. 11.11.6 MS/MS contamination and interferences
11.11.6.1 If the MS/MS mode is to be used without chromatographic separation (e.g., for rapid screening), then the method blank analysis must show that the sample preparation and analysis procedures are free of contamination by the analyte of interest or by interfering compounds. Refer to Method 8000 for guidance on acceptable method blank performance. If contamination is detected in the method blank above acceptable limits, re-extraction and reanalysis of the affected samples are necessary.
11.11.6.2 The MS/MS spectra of a calibration standard and the sample should be compared and the ratios of the three major (most intense) ions
examined. These ratios should be approximately the same unless there is an interference. If an interference appears, chromatographic separation must be utilized.
11.11.6.3 The signal of the target analyte in a sample may be suppressed by co-extracted interferences which do not give a signal in the
monitored ions. In order to monitor such signal suppression, an internal standard may be spiked into all standards, blanks, and sample extracts at a consistent concentration prior to analysis. The internal standard may be any compound which responds well in the appropriate ionization mode and which is not likely to be found in nature. (Atrazine-d5 has been used successfully for positive ion analysis, while 2,6-dinitrotoluene-d3 has been used successfully for negative ion analysis.) The amount spiked should be chosen such that the signal produced is at least 100 times the noise level for the appropriate ion. The signal of the internal standard should be monitored. Reanalysis is necessary for any sample in which the internal standard peak height varies by more than 30% from the average internal standard height obtained during the five-point calibration. If reanalysis confirms this
variance in signal, the sample should be reanalyzed using a chromatographic separation. Quantitation of analyte concentration may be performed using this internal standard. External standard quantitation is also allowed.
11.11.7 The total area of the quantitation ion(s) is calculated and the initial calibration is used to calculate sample results.
11.11.8 MS/MS techniques can also be used to perform structural analysis on ions represented by unassigned m/z ratios. The procedure for compounds of unknown structures is to set up a CAD experiment on the ion of interest. The spectrum generated from this experiment will reflect the structure of the compound by its fragmentation pattern. A trained mass spectroscopist and some history of the sample are usually needed to interpret the spectrum. CAD experiments on actual standards of the expected compound are necessary to confirm or deny that a substance is present.
11.12 Optional wire-repeller CAD confirmation
11.12.1 See Figure 3 for the correct position of the wire-repeller in the thermospray source block.
11.12.2 Once the wire-repeller is inserted into the thermospray flow, the voltage can be increased to approximately 500 - 700 v. Enough voltage is necessary to create fragment ions, but not so much that shorting occurs.
11.12.3 Continue as outlined in Sec. 11.9. 12.0 DATA ANALYSIS AND CALCULATIONS
See Sec. 11.10 and Method 8000 for information on data analysis and calculations.
13.0 METHOD PERFORMANCE
13.1 Performance data and related information are provided in SW-846 methods only as examples and guidance. The data do not represent required performance criteria for users of the methods. Instead, performance criteria should be developed on a project-specific basis, and the laboratory should establish in-house QC performance criteria for the application of this method. These performance data are not intended to be and must not be used as absolute QC acceptance criteria for purposes of laboratory accreditation.
13.2 Single-operator accuracy and precision studies have been conducted using spiked sediment, wastewater, sludge, and water samples. Tables 4, 5, and 6 provide example single-laboratory data for Disperse Red 1. Table 10 provides the data for organophosphorus
pesticides, Table 11 provides the data for tris-BP, Table 12 provides the data for
chlorophenoxyacid herbicides, and Tables 14 and 15 provide the data for carbamates. These data are provided for guidance purposes only.
13.3 Table 13 presents example multi-laboratory accuracy and precision data for the chlorinated phenoxyacid herbicides. Table 13 is based on data from three laboratories that performed replicate analyses of standard solutions at each concentration listed in the table. These data are provided for guidance purposes only.
13.4 Tables 16 and 17 present example multi-laboratory accuracy and precision data for the carbamates. The data summary is based on data from nine laboratories that analyzed triplicate solvent solutions at each concentration listed in the tables. These data are provided for guidance purposes only.
13.5 Table 18 provides example data for the solid-phase extraction of 2,4-D and 2,4,5-TP spiked into TCLP buffers at two different levels. These data are provided for guidance purposes only.
13.6 A single-laboratory study of 31 carbamates and related compounds was conducted in 1998. Bulk quantities of a clay soil and the effluent from a publicly-owned treatment works (POTW) were collected, homogenized, and spiked with the carbamates. The spiking levels were chosen to be approximately 80% of the Universal Treatment Standard (UTS) values for wastewater and non-wastewater, rounded to two significant figures. The spiking levels for the effluents samples are shown in Table 19, and ranged from 22 to 45 µg/L. The spiking levels for the soil samples are shown in Table 20, and ranged from 110 to 4500 µg/kg.
The spiked material was divided into aliquots of suitable size for extraction and analysis. Effluent samples were extracted using continuous liquid-liquid extraction, as described in Method 3520. Soil samples were extracted using Soxhlet extraction, as described in Method 3540. Four aliquots of each sample matrix were prepared and analyzed using all three detector systems in the method. The mean recovery and the relative standard deviation (RSD) of each analyte for the effluent and soil samples are shown in Tables 19 and 20, respectively. These data are provided for guidance purposes only. All data are taken from Reference 11.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any t
